Hundreds of partially overlapping chromosomal fragments could be conceivably rejoined via mechanisms involving no significant DNA synthesis, e.g., by (i) non-homologous end-joining (NHEJ) of DNA fragments1,2 held in register by some special form of chromatin3 or (ii) homologous recombination (HR) via conservative crossovers involving ends of overlapping fragments4-6. Because of the large number of DNA fragments, significant DNA repair synthesis can be involved in (iii) single-strand annealing (SSA) requiring a strand-biased exonucleolytic erosion of the DNA fragments' ends liberating the complementary strands for the annealing with other fragments' complementary single-strand protrusions4.
Alternatively, fragment assembly could require or involve massive DNA synthesis, e.g., (iv) by strand invasion between overlapping fragments (D-loop formation) priming the extension of paired 3′ ends (e.g., by a moving D-loop, like in transcription, FIG. 4) to the end of the templating fragment, followed by the annealing of thus extended ends via complementary single-strand tails (so-called synthesis dependent strand annealing or SDSA mechanism7), or (v) by any kind of “copy-choice” (CC) DNA replication mechanism switching between and copying double-stranded templates from one fragment to another until a full-size chromosome is newly synthesised8.
It is an object of the present invention to form novel hybrid species or novel chromosomes by in vivo intergeneric and/or interspecific recombinations, in a way that is both efficient and easy to reduce to practice.
It is another object of this invention to use in vivo chromosomal engineering, e.g. in an extremophile bacterium Deinococcus D. radiodurans, or any other radiation and/or desiccation and/or chemical treatment resistant organism possessing similar DNA repair mechanisms (the herein described ESDSA) for assembling their fragmented DNA into intact functional chromosomes.
It is still another object of this invention to use in vitro chromosomal engineering, e.g. in D. radiodurans cell-free extracts, or in active cell-free extracts of any other radiation and/or desiccation resistant organism possessing similar DNA repair mechanisms (the herein described ESDSA) for assembling their fragmented DNA into intact functional chromosomes. It is another object of this invention to use the aforesaid strategy to provide sites of integration of exogenous DNA by adding single-stranded or double-stranded terminal additions identical, or similar to, any chromosomal sequence as sites for their integration during the DNA repair process either in vivo or in vitro.
It is another object of this invention to use intact or fragmented DNA from any biological species, or even DNA created by artificial synthesis, as genetic elements to be integrated into the bacterial chromosome and become an integral part thereof.
Still another object of this invention is to use intact or fragmented DNA from any biological species, or even DNA created by artificial synthesis, as genetic elements to be integrated into artificial chromosomes by either in vivo or in vitro chromosomal assembly processes.
It is another object of this invention to use natural transformation capacity, or bacterial sex (e.g., conjugation or transduction), or artificial means (electroporation or chemical modification of the cellular membrane) of introducing foreign DNA into the target cells undergoing DNA repair. This ongoing repair by fragment assembly is triggered by ionizing or ultraviolet irradiation, or by chemicals that damage DNA (e.g., mitomycin C) before or after the introduction of foreign DNA in the bacterial cell. It is another object of this invention to use large genomic or plasmidic DNA fragments containing entire operons for biosynthetic pathways producing (i) useful small molecules or therapeutic proteins, (ii) enzymatic activities for biodegradation or bioremediation of the environment or (iii) energy generating metabolism, for example taking also advantage of the extreme desiccation resistance of D. radiodurans and its innocuousness for humans.
It is still another object of this invention to use a source of biological activity according to this invention as the vector for natural or engineered biodegradation and bioremediation pathways.
It is still another object of this invention to use this chromosomal engineering process in cell free extracts, or with purified proteins, for generating new chromosomal assemblies that can be transferred into appropriate cells, or DNA-free cells, creating new biological entities. These new biological entities or species can be used as biological machines tailored to solve major problems for humanity, e.g., environmental clean-up through biodegradation, synthesis of new therapeutics, clean energy production etc.